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BRIEF REPORT |
Departments of Medicine (H.G., S.R.) and Pediatrics (S.R.) and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Institute of Interdisciplinary Research (J.V.S.), University of Brussels, 1070 Brussels, Belgium; Clalit Health Service (A.H.-D.M.), Um-El Fahem 30010, Israel; Pediatric Endocrine Unit (Y.T.-R.), Ha’Emek Medical Center, Afula 18101, Israel; and Technicon Faculty of Medicine (Y.T.-R.), Haifa 31096, Israel
Address all correspondence and requests for reprints to: Helmut Grasberger, The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: hgrasber{at}uchicago.edu.
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Abstract |
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Objective: We report and study a novel familial TSHR mutation (L653V).
Results: Homozygous individuals expressing L653V had euthyroid hyperthyrotropinemia. Paradoxically, patients had significantly higher 2-h radioiodide uptake and 2- to 24-h radioiodide uptake ratios compared with heterozygous, unaffected family members, suggesting an imbalance between iodide trapping and organification. In transfected COS-7 cells, the mutant TSHR had normal surface expression, basal activity, and TSH-binding affinity, equally (2.2-fold) increased EC50 values for TSH-induced cAMP and IP accumulation, and normal maximum cAMP generation. In contrast, the efficacy of TSH for generating IP was more than 7-fold lower with the mutant compared with wild-type TSHR.
Conclusions: We identified and characterized a TSHR defect, preferentially affecting the IP pathway, with a phenotype distinct from previously reported loss-of-function mutations. Results provide the first in vivo evidence for the physiological role of the TSHR/IP/Ca2+ cascade in regulating iodination. According to systematic in vitro mutagenesis studies, other TSHR mutations can result in even complete loss of IP signaling with retained cAMP induction. We hypothesize that such TSHR mutations could be the cause in unexplained partial organification defects.
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Introduction |
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Biallelic, partial TSHR loss-of-function mutations have been identified in patients with resistance to TSH (RTSH) characterized by congenital hyperthyrotropinemia, normal or low free thyroid hormone (TH) levels, and normal-sized or hypoplastic thyroid gland with low thyroidal radioactive iodine uptake (RAIU) (for review, see Ref. 6). These hallmarks of RTSH were attributed to diminished cAMP signaling capacity. However, the dual activation by TSH of the cAMP and IP/Ca2+ pathways implies that specific loss-of-function TSHR mutations could lead to differential effects and ultimately distinct clinical subtypes of RTSH.
We report a novel TSHR mutation with imbalanced impairment of the cAMP and IP/Ca2+ pathways that could be demonstrated in vitro and in vivo.
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Subjects and Methods |
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The proposita (subject 4) (Fig. 1A
), of Arab-Muslim descent, was born to first cousins. Thyroid function tests (TFTs), performed to evaluate sinus tachycardia, revealed markedly elevated serum TSH (53 mU/liter) but normal free and total TH levels. Antibodies against thyroperoxidase, thyroglobulin, and TSHR were undetectable. Her thyroid gland was in normal position. RAIU was 20% at 2 h (normal, 4–15) and 50% at 24 h (normal, 18–40). Her eutopic thyroid gland had normal size (6 cm3) and echogenicity.
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) also had euthyroid hyperthyrotropinemia (TSH, 49 mU/liter; free T4, 6.8 µg/dl). Her T4 at birth was normal. The oldest sister (subject 1) was markedly hyperthyrotropinemic (TSH, 45 mU/liter). Both sisters had no signs or symptoms of hypothyroidism or hearing impairment. The parents and two other siblings had normal TFTs. TFTs
TFTs were performed as described previously (7). RAIU measurements used a 
scintillation probe. Epithyroid counts were obtained at 2 and 24 h after 2 µCi of oral 131I.
Linkage analysis and DNA sequencing
Studies were approved by the Institutional Review Boards of University of Chicago and Ha’Emek Medical Center. Written informed consents were obtained. Linkage analysis using microsatellite markers (D14S74, D14S606, and D14S616) spanning the TSHR locus was performed as described previously (7). Other markers assessing linkage were D6S960 [0.47 Mbp p-ter of IYD (DEHAL1)], D15S659 (0.96 Mbp q-ter of DUOX2), and intronic single nucleotide polymorphisms within DUOX2, TPO, and PDS. The complete TSHR coding sequence and intron-exon boundaries were sequenced bidirectionally. The c.1957C > G TSHR mutation was confirmed by MlnI digestion.
Construction of expression vectors, cell culture, and transient transfection
The L653V TSHR expression vector was constructed by site-directed mutagenesis of the wild-type (WT) TSHR cloned in plasmid pSVL. COS-7 cells were cultured and transfected as reported previously (8).
Flow immunocytofluorometry
Cell surface expression of TSHR was quantified by flow immunocytofluorometry with monoclonal antibodies 3G4 (9).
Determination of cAMP production
cAMP was measured after 1-h incubation in Krebs-Ringer HEPES with 25 µM rolipram and various concentrations (0–100 U/liter) of bovine TSH (bTSH) (Sigma, St. Louis, MO) as described previously (8).
IP measurements
Cells labeled for 24 h with 20 µCi/ml [3H]inositol were preincubated for 30 min with 10 mM LiCl and then were exposed for 15 min to various concentrations of bTSH. 3H-labeled IPs were extracted and separated by stepwise anion exchange chromatography (3).
Homologous competitive TSH binding assay
Binding of [125I]TSH (58 µCi/mg, 50–60 U/mg; Brahms Diagnostica, Berlin, Germany) to intact cells was determined in the presence of various concentrations of unlabeled bTSH (8).
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Results |
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). All family members shared the same household in a nonendemic area in northern Israel. The high early (2 h) RAIU, indicating increased iodide trapping, prompted testing of all family members, revealing significantly higher 2-h uptake rates and 2- to 24-h uptake ratios in the three affected siblings compared with the normothyrotropinemic subjects (Fig. 1C). We hypothesized that a single recessively inherited defect could cause both euthyroid hyperthyrotropinemia and increased iodide trapping, a finding otherwise associated with defects in iodine organification or iodide deficiency.
Linkage of the phenotype to the TSHR locus was suggested because only affected individuals were homozygous for the same haplotype (Fig. 1B). Linkage to DUOX2, TPO, PDS, or IYD (DEHAL1) was excluded. All affected siblings carried a C to G transversion (c.1957C > G) on both TSHR alleles, producing a novel missense mutation (L653V) in the center of the third extracellular loop (ECL3) of the TSHR heptahelical domain. Both parents and the two nonaffected siblings were heterozygous for this mutation.
We assessed the function of L653V TSHR in transfected COS-7 cells. WT TSHR and P162A TSHR, which exhibits reduced TSH binding affinity (10), were analyzed in the same experiments. Results are summarized in Table 1, and representative experiments are displayed (Fig. 2). L653V TSHR was expressed at the cell surface at almost the same level as WT TSHR, determined in both TSH binding and flow cytometry studies, and had the same affinity for TSH as the WT TSHR (Fig. 2A). As indicated by a right shift of the concentration-response curves relative to those of WT TSHR (Fig. 2, B and C), the EC50 (for bTSH) of the L653V TSHR is higher for the cAMP signal (2.2-fold higher than WT EC50) and IP signal (2.2-fold), similar to the P162A TSHR (2.7-fold for cAMP and 2.8-fold for IP). Thus, the L653V and, as expected, the P162A mutation affect the potencies of bTSH for cAMP and IP induction equivalently. However, the most striking finding was the reduced efficacy of the L653V TSHR for IP induction, measured as IP accumulation above pSVL-transfected cells at 100 U/liter bTSH. Efficacy for IP accumulation was only 14% of WT TSHR level. For comparison, the maximum effect of P162A TSHR (80% of WT) was compatible with its slightly reduced surface expression. In contrast to the mutation-specific effects on IP generation, the maximum cAMP response of WT and mutant TSHRs were essentially identical.
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Discussion |
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In human thyrocytes, the Gs/cAMP pathway controls positively cell proliferation, iodide uptake, and TH secretion, whereas the Gq/IP pathway stimulates iodination and, thus, TH synthesis (5, 11). Sodium iodide symporter expression is the most sensitive biomarker for stimulation of the cAMP cascade by TSH (12, 13). Increased iodide trapping in our patients implied that the elevated TSH levels overcompensate the defect in TSHR signaling. This also suggested that TH synthesis in the patients is limited by a defect in a cAMP-independent pathway, which drives the serum TSH above the level needed for compensation of the mild defect in cAMP signaling. This second pathway is likely an IP-triggered Ca2+ signal known to stimulate the generation of H2O2 crucial for all thyroid peroxidase-catalyzed reactions. We therefore hypothesized that the L653V mutation results in a "pathway-specific" defect, by causing a more severe defect in IP than in cAMP induction.
The importance of individual ECL3 residues for signal transduction was examined recently by a systematic alanine scan (14). Specifically, the L653A TSHR was shown to have essentially normal TSH binding affinity, slightly reduced cell surface expression (85% of WT TSHR), and reduced basal and stimulated cAMP accumulation in transfected COS-7 cells (62% of WT TSHR in stimulated cells). However, TSH-stimulated IP production of the L653A mutant was only 12% of WT TSHR. These data closely resemble our findings for the L653V mutation (Table 1). In addition, we have shown that L653V reduces the potency of TSH for cAMP and IP induction to essentially the same extent, whereas the imbalance in dual-pathway stimulation by the L653V mutant is attributable to diminished efficacy for IP generation.
These results can be explained by the emerging concept of TSHR activation based on an allosteric model (15) in which conversion of the ectodomain from a tethered inverse agonist to an agonist (16) acts as a trigger, facilitating conversion into active conformations. The latter are likely distinguished by differential affinity to couple to Gs or Gq, as proposed for other G protein-coupled receptors interacting with distinct G protein classes and as demonstrated in TSHR-expressing CHO cells (17). In the framework of these models, L653V appears to have two effects that together account for its functional characteristics. First, L653V interferes with a trigger mechanism common to the Gs- and Gq-initiated pathways, as indicated by equivalently reduced potency for cAMP and IP induction similar to an upstream defect in ligand binding (P162A). This interpretation supports the important role of L653V in the intramolecular signal transduction mechanism, specifically in the agonist-like interaction of the ectodomain with the ECLs. Second, on activation of the trigger, L653V has a higher tendency, compared with WT TSHR, for transition into an allosteric state coupling to Gs, i.e. the L653V mutation disturbs the equilibrium between Gs- and Gq-coupled activated receptor states. Thus, an isomerization defect in a model with at least two active allosteric states can explain the drastically diminished efficacy of L653V TSHR for IP induction.
It should be emphasized that the substantially higher EC50 for IP vs. cAMP of WT TSHR in vitro are not caused by distinct potencies to couple to Gs and Gq but rather by differential efficiency of G proteins to activate their downstream effectors (1). G protein-effector coupling depends on cellular context and is modulated by external factors, e.g. by costimulation of Gi-coupled receptors, leading to sensitization of the IP and concomitant inhibition of the cAMP pathway (18). Thus, low sensitivity of the IP pathway for stimulation by TSH in vitro does not preclude a physiological role of this pathway in thyrocytes in situ.
Overall, our results revealed a specific TSHR defect, preferentially affecting the IP pathway, with a different phenotype than reported previously for TSHR inactivating mutations. This is the first in vivo evidence for the physiological role of the TSHR/IP/Ca2+ cascade in regulating iodination. The absence of spontaneous iodide discharge may reflect the limited role of this pathway in stimulating iodination, but one has to consider that the effect of the L653V mutation is neither absolutely specific for the IP pathway nor does it cause a complete loss of IP signaling. The results from in vitro mutagenesis studies indicate the potential for other TSHR mutations to cause similar or even more pronounced and specific IP signaling defects than the L653V mutation (14, 19, 20). Such TSHR mutations could underlie unexplained partial organification defects.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online April 24, 2007
Abbreviations: bTSH, Bovine TSH; ECL3, third extracellular loop; IP, inositol phosphates; RAIU, radioactive iodine uptake; RTSH, resistance to TSH; TFT, thyroid function tests; TH, thyroid hormone; TSHR, TSH receptor; WT, wild type.
Received February 16, 2007.
Accepted April 13, 2007.
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